In Phase II we will continue the development of RainDance Technologies droplet-based microfluidic instrument platform, based on the results from the Phase I proof-of-principle experiments, and develop a commercial system that includes the ability to screen compound libraries in droplets. Our goal by the end of Phase II will be to provide a complete instrument platform able to screen a compound library composed of up to 100,000 members using assays for both a recombinant enzyme and an established cell line. Initial efforts will focus on compound management and handling of barcoded droplets, additional effort will fully enable microfluidic assays and disposable chip designs, and we will complete the integration and engineering development for commercialization before the end of the two year grant period. We believe that significant value to the research and pharmaceutical communities will result from complete development of a droplet-based open platform, specifically for the use of high throughput screening (HTS) formatted droplet libraries with fluorescent in vitro protein and cellular assays. PUBLIC HEALTH RELEVANCE: Miniaturization of laboratory operations in biology and chemistry has reduced reaction volumes from a few milliliters (in test-tubes) to a few microliters (in microtiter plates) - a 1000-fold reduction. The drive to increase throughput by assay miniaturization has been particularly important for drug screening. Today, many industrial screening programs process 100,000 compounds a day (~1 per second), a thousand times as many as were processed in an entire week in 1990. Although robotic microtiter plate based infrastructure has enabled the screening of collections exceeding one million small molecules, the primary screening methodology has largely been restricted to assaying a single high concentration of each compound. Further reducing test volumes below the 1-2 <l capacity of 1536-well plates would create both significant cost savings and enable higher throughput. Furthermore, a primary screen that included a dose-response curve for every compound, for example, would significantly reduce the number of false positives and false negatives compared to a single-point screen, and are able to identify subtle pharmacologies such as partial agonism or antagonism. In addition, it should also be possible to screen each compound simultaneously against multiple targets to determine specificity. However, using microtiter plate technology for further miniaturization is problematic: for example, evaporation becomes significant in microliter volumes, capillary action causes "wicking" and bridging of liquid between wells, and this would not alleviate the time and consumables cost associated with robotic microtiter plate handling. The key technology in this proposal is RainDance Technologies'droplet-based microfluidic platform. Using this platform, we believe we can further reduce the volume of each assay by 103- to 106-fold and increase the throughput >1,000 times, by confining each assay in a separate microscopic droplet. We will use this technology to enable high throughput screening for molecular and cell-based assays, significantly reducing the cost, time, and reagents for screening, and enabling the prospect of high throughput primary cell screening.